Anti-metastasis Effect Of Heparin Derivatives Via Inhibition Of Elevated Mmps

Cancer is a serious threat to human life and health because of the high mortality.One of the important causes is the invasion and metastatic characteristics.Tumor metastasis is a highly controlled process assoiated with multiple factors and links.Degradation of ECM and BM is the important event of the invasion and metastasis.Matrix metalloproteinases(MMPs)are a family of Zn2+ dependent endopeptidases.They are capable to degrade the ECM and BM,which consist of different collagens and other proteins such as proteoglycans.Laminin and fibronectin.Several MMPs,such as MMP-2,MMP-9,play a important role in the degradation of ECM and BM,thus have been associated with tumor growth and invasion.MMPs are a family of zinc-dependent endopeptidases.These enzymes can degrade almost all kinds components of the ECM and BM.The expression level of MMPs is usually low in nomal tissues,but studys found that it’s always abnormally elevated in the process of tumor development.A variety of growth factors can regulate the level of expression of MMPs,wherein TGF-β-mediated signaling pathway on the regulation of MMPs expression plays an important role and gets more and more attention.To inhibit the activity of MMPs,a lot of hMMP inhibitors were prepared.In general,MMPs inhibitors can be divided into five categories-Peptidomimetics,Nonpeptidomimetics,Natural MMPIs,Tetracycline-like derivatives and Bisphosphonates.In recently years,polysaccharide has also become the important sources of antitumor drug.Heparin derivatives M402 is typical sucessfull examples.By periodate oxidation/sodium borohydride reduction,we got a glycol-splitting heparin derivative named HP-sp.In order to study the antitumor activity of HP-sp,HP and M402 were taken as control.The study included structure confirmation of heparin derivatives,evaluation of anti-transferation avtivity in vitro and antitumor transfer activity of heparin derivatives in vivo.The results and conclusions of the research are as follows:1.The preparation of heparin derivativesBy periodate oxidation/sodium borohydride,we got the heparin glocol-splitting derivatives.Then We applied high performance size exclusive chromatography method to determine the molecular weight of HP-sp.The determination results were as follows:HP,Mn=14037,Mw=17153,Mz=20697;HP-sp,Mn=10697,Mw=13254,Mz=17261.2.The antitumor activity in vitro of HP,HP-sp,M4022.1 MTT assayMTT assay was used to detect the influence of HP,HP-sp,M402 on tumor cell proliferation.The result showed that HP,HP-sp,M402 had no significant inhibitory effect on tumor proliferation.2.2 Scratch assayScratch assay was used to detect the effect of HP,HP-sp,M402 on cell migration.The research showed that HP,HP-sp,M402 can significantly inhibit the migration of HepG2 cells with time and dose dependency.2.3 Transwell assayTranswell assay was used to detect the effect of HP,HP-sp,M402 on cell migration.The results are similar with Scratch assay.HP,HP-sp,M402 can significantly inhibit the migration of HepG2 cells with time and dose dependency.2.4 Matrigel assayMatrigel assay was used to detect the effect of HP,HP-sp,M402 on cell invasion.The research shows that comparing to Negative,MMPI,HP,HP-sp,M402 can significantly inhibit the invasion of HepG2 cells.Moreover,HP,HP-sp,M402 show greater inhibition of invasion comparing to MMPI.2.5 Western Blot assayWestern Blot assay was used to detect the effect of HP,HP-sp,M402 on the expression of MMP-2,-9.The research shows that HP,HP-sp,M402 can significantly decrease the expression of MMP-2,-9.Moreover,HP,HP-sp,M402 can also significantly decrease NF-κβand p38 expression levels,which involving the TGF-β-mediated signaling pathway.3.The antitumor activity in vivo of HP,HP-sp,M402Using the mouse liver cancer model,we evaluated activity of HP,HP-sp,M402 to inhibit tumor metastasis in vivo.Compared with DEN+NMOR group,the HP,HP-sp,M402 could significantly inhibit the progress of liver cancer.Immunohistochemical results showed that comparing to DEN+NMOR group,the expression of MMP-2,-9 of HP,HP-sp,M402 were decreased significantly.HE staining results showed that in the DEN+NMOR group,a large number of cells in the liver necrosis and with the liver congestion.Comparing to DEN+NMOR group,HP,HP-sp,M402 significantly improve the live condition.In conclusion,HP,HP-sp,M402 can inhibit the invasion and metastasis of tumor invitro and vivo,considering the side effects of HP,HP-sp is expected to be developed as a new generation of polysaccharide antitumor drugs.